Activating mutations of the rejceptor tyrosine kinase FLT3 are one of the most common mutations found in acute myeloid leukemia (AML) and are clearly associated with a poor prognosis. FLT3 therefore represents a logical therapeutic target. During the first five years of this project, we used in vitro studies to identify and characterize a series of small molecule FLT3 inhibitors for use as monotherapy and in combination with chemotherapy, and carried out early phase single agent trials of the most promising compounds. Over the past five years we carried out correlative studies using samples collected from a large number of trials of FLT3 inhibitors used as single agents and in combination with chemotherapy. We have determined that potent FLT3 inhibition in vivo results in significant clinical benefit, inducing clearance of circulating leukemia cells and terminal differentiation of marrow blasts. We hypothesize that survival for patients with FLT3/ITD AML can be significantly improved with the use of FLT3 inhibitors. For younger patients, we predict that FLT3 inhibition combined with induction chemotherapy will increase the remission rate, and that combination with consolidation therapy, as well as maintenance therapy, will minimize the relapse risk. For older patients, the combination of FLT3 inhibition with hypomethylating agents offers the potential for synergistic anti-leukemic effect as well as better tolerability. However, the data from our correlative studies indicates that a number of clinical variables influence the efficacy of FLT3 inhibition, depending on the context in which the TKIs are used. Monotherapy with FLT3 TKIs leads to the development of resistance mutations, while the combination of FLT3 TKIs with chemotherapy is complicated by the effects of c-KlT inhibition, the competing effects of FLT3 ligand, and less predictable pharmacokinetics. In order to address these issues, we propose to optimize the use of two FLT3 TKIs, AC220 and crenolanib, with a combination of in vitro studies and clinical trials with correlative studies. Our approach is organized into 3 specific aims. First, we will establish the optimal dose and schedule for AC220 both as monotherapy and in combination with standard chemotherapy using correlative studies from clinical trial samples. Second, we will investigate combinations of FLT3 inhibitors with DNA methyltransferase inhibitors (DNMTi), both in vitro and in the context of a proposed clinical trial. Third, we will use in vitro cell line models and primary AML samples cultured with bone marrow stroma, as well as correlative data from a proposed phase 2 clinical trial, to establish the efficacy of crenolanib as a FLT3 inhibitor with activity against FLT3 kinase domain mutations. RELEVANCE (See instructions): The translational impact of the proposed work will be to synthesize the laboratory correlative data obtained from several previous monotherapy and combination FLT3 inhibitor trials into a series of focused trials designed to improve outcomes for specific subsets of patients with FLT3/ITD AML. The clinical impact of this proposed work will be to provide exact treatment regimens, derived from an integration of FLT3 inhibitors into standard-of-care practice, that will improve the overall survival for these patients.